Anal. Chem. 1998, 70, 3946-3951
An Amperometric Urea Biosensor Based on a Polyaniline-Perfluorosulfonated Ionomer Composite Electrode Wen-June Cho and Hsuan-Jung Huang*
Department of Chemistry, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
A new procedure for urea determination was developed. By cross-linking urease onto a polyaniline-Nafion composite electrode which sensed the ammonium ion effectively, a very sensitive urea biosensor was formed. The effects of applied potential, pH of buffer solutions, flow rate of carrier, and possible interferences on the response of urea biosensor were studied. With the developed urea biosensor, a detection limit as low as 0.5 µM and a response time as short as 40 s were obtained in an flow injection analysis system. A relative standard deviation of 2.2% (n ) 15) was obtained for the successive analysis of a 0.03 mM standard urea solution. Applicability of the urea biosensor for urea analysis was demonstrated by the analysis of NIST standard reference material and urine samples. Since the first urea biosensor was prepared by Guilbault et al.,1 the use of urease as a biocatalyst for the development of urea biosensors has attracted continuous interest from biochemical and clinical analysts, and various types of urea biosensors have thus been reported.1-25 To fabricate a urea biosensor, the urease was (1) Guilbault, G. G.; Smith, R. K.; Montalvo, J. G. Anal. Chem. 1969, 41, 600. (2) Liu, D.; Ge, K.; Cheng, K.; Nie, L.; Yao, S. Anal. Chim. Acta 1995, 307, 61. (3) Mascini, M. Sens. Actuators, B 1995, 29, 121. (4) Xie, X.; Suleiman, A. A.; Guilbault, G. G. Talanta 1991, 38, 1197. (5) Adeloju, S. B.; Shaw, S. J.; Wallace, G. G. Anal. Chim. Acta 1993, 281, 611. (6) Adeloju, S. B.; Shaw S. J.; Wallace, G. G. Anal. Chim. Acta 1993, 281, 621. (7) Esteve, M. F.; Alegret, S. J. Chem. Educ. 1994, 71, A67. (8) Liu, D.; Meyerhoff, M. E.; Goldberg, H. D.; Brown, R. B. Anal. Chim. Acta 1993, 274, 37. (9) Ivnitskii, D. M.; Rishpon, J. Anal. Chim. Acta 1993, 282, 517. (10) Walcerz, I.; Koncki, R.; Leszczynska, E.; Glab, S. Anal. Chim. Acta 1995, 315, 289. (11) Liu. C. H.; Bao. Y. F.; Deng, J. Q. Prog. Biochem. Biophys. 1995, 22, 555. (12) Caras, S. S.; Janata, J. Anal. Chem. 1980, 52, 1935. (13) Hanazato, Y.; Nakako, G.; Maeda, M.; Shiono, S. Anal. Chim. Acta 1987, 193, 87. (14) Vering, T.; Schuhmann, W.; Schmidt, H. L.; Mikolajick, T.; Falter, T.; Ryssel, H.; Janata, J. Electroanalysis 1994, 6, 953. (15) Chen, K.; Liu, D.; Nie, L.; Yao, S. Talanta 1994, 41, 2195. (16) Jdanova, A. S.; Poyard, S.; Soldatkin, A. P.; Jaffrezic-Renault, N.; Martelet, C. Anal. Chim. Acta 1996, 321, 35. (17) Sangodkar, H.; Sukeerthi, S.; Srinivasa, R. S.; Lal, R.; Contractor, A. Q. Anal. Chem. 1996, 68, 779. (18) Adams, R. E.; Carr, P. W. Anal. Chem. 1978, 50, 944. (19) Nikoleis D. P.; Siontorou, C. G. Anal. Chem. 1995, 67, 936. (20) Petersson, B. Anal. Chim. Acta 1988, 209, 239. (21) Osborne, M. D.; Girault, H. H. Electroanalysis 1995, 7, 714. (22) Rui, C. S.; Kenji, S.; Kato, Y. Anal. Sci. 1992, 8, 845. (23) Guilbault, G. G.; Seo, M. L. Talanta 1994, 41, 1029.
3946 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
immobilized onto a membrane or support in which the urea was catalytically converted into ammonium and bicarbonate ions. A transducer was then employed to monitor the ions produced by the enzymatic reaction. For monitoring the enzymatic products, various techniques, such as spectrometry,2-4 potentiometry with the application of a pH-sensitive electrode, an ammonium ion selective electrode, and an ammmonium ion-sensitive field effect transistor,5-14 conductometry,15-17 coulometry,18 and amperometry,19-25 have been proposed. Despite the various developments proposed, the key parameter for producing a useful urea biosensor rests on the implantation of a sensitive and reliable transducer that transduces the enzymatic reaction products into detectable signal. Among the transducers developed, potentiometric and amperometric determinations are the two transduction methods frequently used. A potentiometric transducer that monitors the potential changes due to the generation of ammonium or bicarbonate ions with an ion-selective electrode is no doubt the most commonly used technique. But it suffers from the inherent characteristics of potentiometry, i.e., rather sluggish response to solutions of low analyte concentration, vulnerability to the interferences of other ions in sample solutions, and relatively high detection limit. For amperometric determination, systems with an additional enzyme, e.g., glutamate dehydrogenase,21-23 or with the application of conducting polymer-modified electrodes24,25 were developed. Amperometric determination shows better sensitivity and lower detection limit compared with those obtained by potentiometric methods. In this report, a new procedure for urea determination has been developed. Urease was immobilized directly onto a polyaniline (PA)-Nafion composite electrode which sensed the ammonium ions effectively and formed a very sensitive urea biosensor. Characteristics of high sensitivity, lower detection limit, and short response time were found for the proposed urea biosensor. EXPERIMENTAL SECTION Preparation of the PA/NF/UR Electrodes. Electrochemical polymerization of PA/Nafion film was carried out according to the previous report.26 A 4.0-µL portion of 2.0 wt % Aldrich Nafion 117 [equivalent weight 1100, dissolved in ethanol-water (9:1)] (24) Trojanowicz, M.; Lewenstam, A.; Krawczyk, T. K. V.; Lahdesmaki, I.; Szczepek, W. Eletroanalysis 1996, 8, 233. (25) Aadeloju, S. B.; Shaw, S. J.; Wallace, G. G. Anal. Chim. Acta 1996, 323, 107. (26) Sung, J. Y.; Huang, H. J. Anal. Chim. Acta 1991, 246, 275. S0003-2700(98)00004-3 CCC: $15.00
© 1998 American Chemical Society Published on Web 08/14/1998
solution was applied onto a glassy carbon disk electrode (3 mm diameter) and air-dried to allow the solvent to evaporate. The polyaniline film was deposited by immersing the electrode in a solution containing 0.1 M aniline and 1.0 M sulfuric acid and sweeping the potential between -200 and 800 mV (vs Ag/AgCl reference electrode) with a sweep rate of 20 mV s-1 for 10 min (about 45-48 cycles). The amount of charges for the polyaniline deposited was controlled at about 2.4 × 10-3 C. The finished electrode shows a very smooth surface and shines with a light green color. The PA/Nafion-coated electrode was rinsed with pure water several times to remove the aniline and sulfuric acid residues on the electrode surface. It was conditioned in a pH 6.50 phosphate buffer solution by applying a potential of -200 mV for 30 min in a flow injection analysis (FIA) system before use. The cross-linking procedure was used for the immobilization of urease on the PA/Nafion electrode. The urease solution used for immobilization was prepared by dissolving 200 mg of urease (33 units mg-1) in 5 mL of 0.5% (w/v) bovine serum albumin (BSA) solution. A 2% glutaraldehyde (GA) solution was used as the cross-linking agent. The prepared urease solution and the 2% GA solution in a volume ratio of 20:6 were then mixed to form the urease immobilization solution (with an activity of 1015 units/ mL). After an appropriate amount (unless otherwise specified, 26 µL) of the urease (UR) immobilization solution was applied on the conditioned PA/Nafion (NF) electrode, it was allowed to airdry to form a PA/NF/UR electrode with a urease activity of 26.4 units. The finished PA/NF/UR electrode was stored at 4 °C in a pH 6.5 phosphate buffer solution when not in use. For electrodeposition and electrochemical studies, the threeelectrode system was used. A piece of Pt wire and a Ag/AgCl (with 3 M NaCl solution) electrode were used respectively as the counter and reference electrodes. The electrodeposition was performed with a PAR 175 universal programmer coupled with a PAR 179 coulometer. For cyclic voltammetric and amperometric measurements, the system including a flow-through thin-layer electrochemical cell (BAS LC-17A, with the PA/Nafion or PA/ NF/UR coated on glassy carbon electrode) connected with a BAS 100A electrochemical analyzer was used. FIA and HPLC Systems. The FIA measurements were conducted with a Gilson Minipuls 3 peristaltic pump, a Rheodyne 7125 injector with a 100-µL sample loop, and the PA/NF/URcoated flow-through thin-layer electrochemical detector. In the HPLC measurement, the flow of solutions was controlled by a Spectra-Physics isocratic pump (SP8810) having a 4.6-mm i.d. × 250-mm stainless steel column packed with ODS2 silica (Fisons 5-µm particle size) as the separation column. The mobile phase used for ammonium and urea separation was a pH 6.5 phosphate buffer solution. The solution was filtered through a 1.0-µm filter prior to use. The flow rate was controlled at 0.5 mL/min throughout the experiment. Reagents. Lyophilized Jack bean urease (EC 3.5.1.5.), type IX, with a specific activity of 33 units mg-1 lyophilisate, bovine serum albumin (96-99%), and β-D-(+)-glucose (97%) were obtained from Sigma Chemical Co. (St. Louis, MO). Urea, uric acid (99.9%), L-ascorbic acid (99%), glutaraldehyde (25% solution), and Nafion (5 wt %) were obtained from Aldrich (Milwaukee, WI). The SRM 912A urea was obtained from NIST, Gaithersburg, MD.
Figure 1. Cyclic voltammograms recorded during electrochemical deposition of polyaniline at a Nafion-coated electrode in solutions containing 0.1 M aniline and 1.0 M sulfuric acid. Potentials were swept between -200 and 800 mV (vs Ag/AgCl) with a sweep rate of 20 mV s-1.
All chemicals and solvents used were of analytical grade and were used as received. The phosphate buffer solutions were prepared from 0.10 M sodium dihydrogen phosphate with the addition of an appropriate amount of 0.1 M NaOH solution. Deionized-RO water prepared from a Milli-Q system (Millipore) with R no less than 16 MΩ-cm was used for preparing the solution. RESULTS AND DISCUSSION Characteristics of PA/Nafion Electrodes. The CVs obtained during the electrodeposition of polyaniline are shown in Figure 1. They are similar to that reported in the literature.27-31 The three pairs of redox peak indicate the presence of discrete electroactive regions in the film. The shape of the voltammograms varies with the experimental conditions of electrodeposition, in both the relative intensities and the position of peaks. From literature, the mechanism of the redox reactions of polyaniline can be summarized as illustrated in Scheme 1.27-31 The equilibria between structures B, C, and D represent the first-stage oxidation-reduction and the accompanying proton elimination-addition process, while the equilibrium between C and F is the anion doping-undoping process. The redox pair with peak potentials at 0.10 and 0.21 V shown in Figure 1 was related to these reactions. The equilibria between structures D, E, and G represent the redox processes of polyaniline in proton-deficient solutions. They are the second-stage redox reactions. Because of the deficit of protons, doping-undoping of anions becomes the dominant process. Redox couples with peak potentials at 0.68 and 0.78 V were related to these reactions. The redox pair with peak potentials at about 0.57 V was suggested to be due to the higher oxidation level of aniline. It occurs when the applied potential is (27) Genies E. M.; Tsintavis, C. J. Electroanal. Chem. 1986, 200, 127. (28) Genies E. M.; Tsintavis, C. J. Electroanal. Chem. 1985, 195, 109. (29) Kitani, A.; Izumi, J.; Yano, J.; Sasaki, K. Bull. Chem. Soc. Jpn. 1984, 57, 2254. (30) Orata, D.; Buttry, D. A. J. Am. Chem. Soc. 1987, 109, 3574. (31) Genies, E. M.; Lapkowski, M. J. Electroanal. Chem. 1987, 200, 67.
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Scheme 1
Figure 2. Current response of the PA/Nafion electrode for the injection of 1 mM to ∼0.01 mM NH4+ (a) and urea (b) solutions in a pH 6.5 phosphate buffer solution. A potential of -200 mV was applied on the PA/Nafion electrode. Flow rate used was 4.5 mL/min.
larger than 0.70 V. It may be attributed to the conversion of a benzenoid-like to the quinoid-like polymeric structure with an ortho coupling of aniline or to the degradation of polymeric structure.27,31 It has been demonstrated that, in the presence of Nafion, which is a cation exchanger, the immobilized sulfonate groups of Nafion serve as a charge compensator in the course of anodic polymerization of polyaniline and polypyrrole.32-34 The diffusing species accompanying the redox processes of Nafion-coated polyaniline or polypyrrole films were shown to be cations as opposed to anions in the case of plain polyaniline or polypyrrole films.33-35 It is therefore feasible to use Nafion as a modifier of polymer-coated electrodes; i.e., in the presence of immobilized sulfonate groups, the movement of anions into the polymer-Nafion composite layer will be hindered while the movement of cations becomes necessary to accomplish the redox process of the polymer. In a flow system, if the polymer-Nafion composite electrode is poised at an appropriate potential to effect reduction of the polymer, a transient current will occur whenever a sample plug containing cations arrives at the composite electrode. Although the transient current is due to the reduction of polymer, its magnitude will be proportional to the cation concentration in the sample plug. Response of PA/Nafion Electrode to NH4+ in FIA Determination. The capability of the PA/Nafion electrode as a detector for NH4+ was demonstrated by the injection of NH4+ solutions into a FIA system. Figure 2a shows the responses obtained upon (32) Fan, F.-R. F.; Bard, A. J. Electrochem. Soc. 1986, 133, 301. (33) Nagasubramanian, G.; Die Stefano, S.; Moacanin, J. J. Phys. Chem. 1986, 90, 4447. (34) Hirai, T.; Kuwabata, S.; Yoneyama, H. J. Electrochem. Soc. 1988, 135, 1132. (35) Chang, C. M.; Huang, H. J. Anal. Chim. Acta 1995, 300, 15.
3948 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
Figure 3. Effect of pH on the current response of the PA/NF/UR electrode. Solutions containing 1.0 mM urea in phosphate buffer of different pH values were used, and the flow rate was controlled at 0.45 mL/min. A potential of -200 mV was applied on the PA/NF/UR electrode.
the injection of 0.01-1.0 mM NH4+ solutions. A pH 6.5 phosphate buffer solution was used as the carrier, and a potential of -200 mV was applied on the PA/Nafion electrode. For comparison, urea solutions with the same concentration as those of NH4+ were analyzed (Figure 2b). Much smaller responses from the hydrolysis of urea to urenium ions were found for the analysis of urea solutions. The variation of current responses to the change of applied potential on the PA/Nafion electrode was also studied. With the injection of 1 mM NH4+ solutions in an applied potential
range from -300 to 200 mV, a current maximum was found at -200 mV. The current response decreased rapidly as the applied potential increased to -100 mV. It approached zero as the applied potential became larger than 0.000 mV. The current response at -300 mV was found to be only about half of that obtained at -200 mV, and its stability deteriorated. An applied potential of -200 mV was thus adopted as an optimum potential throughout the whole experiment. Characterization of the PA/NF/UR Electrode. Detection by the PA/NF/UR electrode requires the urea molecules to diffuse to the electrode surface and undergo the enzymatic reaction to yield NH4+ and HCO3- ions. The NH4+ diffuses further to the PA/Nafion film and triggers the reduction of polyaniline on the electrode. As ammonium and bicarbonate ions are the products of urease reaction in a phosphate media,36,37 the reactions occurring at the PA/NF/UR electrode can be expressed by the following equations after referring to Scheme 1 and considering the effect of Nafion on polyaniline electrode discussed above. urease
urea 9 8 NH4+ + HCO3HO
(1)
NH4+ + PA+‚RSO3- + e- f PA‚NH4+‚RSO3-
(2)
2
where PA+ and PA represent the oxidized and reduced forms of polyaniline and RSO3- represents the skeleton of Nafion with the immobilized sulfonate groups. Equation 2 represents the cation doping process accompanying the reduction of PA at the PA/ Nafion electrode. From eq 2, whenever an NH4+ ion is produced and doped into the PA/Nafion film, the flow of reduction current will be induced. Optimization for FIA Determinations. The pH effect on the response of the PA/NF/UR electrode was studied by varying the pH of the 0.1 M phosphate buffer solutions from 6.00 to 7.75. Figure 3 shows the plot of the responses of the PA/NF/UR electrode to the injection of solutions of 1.0 mM urea at different pH values. The highest current response was found for solutions with a pH of 7.50. Because the polyaniline shows better reactivity in more acidic solution, 0.1 M phosphate buffer solution with a pH of 6.50 was chosen as the optimal carrier solution for urea analysis. The effect of the flow rate of the carrier on the current response was also studied. For flow rates ranging from 0.36 to 0.63 mL/ min studied, the current response decreased gradually as the flow rate increased. Because a higher current response was obtained at the expense of analysis time, a compromise value of 0.45 mL/ min was selected as the optimal flow rate for this experiment. The response time of the PA/NF/UR electrode for urea analysis was studied for the injection of solutions containing from 1.0 mM to 1.0 µM of urea. In the concentration range studied, the response time remained essentially constant with the change of urea concentration in solution. In the studied solutions, it took about 2 min for the reduction current to go through its maximum and return to the background level. As the response of a PA/ Nafion electrode to the doping of NH4+ is very fast, the response time of a PA/NF/UR electrode should depend on factors involved (36) Jespersen, N. D. J. Am. Chem. Soc. 1975, 97, 1662. (37) Buhl, S. N.; Jackson, K. Y.; Lubinski, R.; Vanderlinde, R. E. Clin. Chem. 1976, 22, 1872.
Table 1. Current Response and Response Time for PA/NF/UR Electrodes Prepared by Applying Different Amounts of Urease Immobilization Solution (with Urease Activity of 1.01 units/µL)
amont of urease immobilization solution applied (µL) 26.0 13.0 7.5 4.0
current response (nA) to solutions of various urea concentration 1 mM 0.1 mM 0.01 mM 704 593 540 394
82 80 64 63
response time (s)
6.1 5.9 5.7 5.5
130 100 80 40
in the analyzing processes. The movement of urea molecules across the barrier of the BSA and GA layer on the electrode surface and the diffusion of NH4+ to the PA/Nafion film may induce a substantial increment of response time. It was found that the response time can be effectively decreased by applying less urease immobilization solution on the PA/Nafion electrode. Tabel 1 shows the current response and the response time for PA/NF/UR electrodes prepared by applying various amount of urease immobilization solution. From Table 1, the response time can be decreased to as little as 40 s with electrodes prepared by applying 4 µL of the urease immobilization solution. The response times obtained in this experiment are shorter than the response time of 2-5 min reported in the literature.10,22-25 Reproducibility and Stability of the PA/NF/UR Electrodes. The reproducibility of the PA/NF/UR electrodes was studied by making successive injections of the 0.03 mM standard urea solutions in the FIA system. The relative standard deviation (RSD) for this analysis was 2.22% (n ) 15). The rather low RSD value obtained proved the feasibility of using the urease electrode for repetitive applications. The dynamic range of the urea electrode was investigated by monitoring the standard urea solutions in a concentration range from 1.0 µM to 10 mM. Figure 4a shows a plot of the current response versus the concentration of standard urea solutions. Good linearity was found in the low concentration range of the calibration graph. A correlation coefficient of 0.9995 was obtained for the linear calibration plot in the concentration range of 1.0-1000 µM (shown as the inset in Figure 4a). When the urea concentration was larger than 1.0 mM, a curvature on the calibration graph appeared, and the current response started to level off as the urea concentration was further increased. The sluggish increment of current response for solutions of higher concentration should be due to the kinetic restriction of the enzymatic reaction involved.38 When the concentration of substrate was overloaded, the original first-order enzymatic reaction would have been changed to a zeroth-order reaction on which the reaction rate became independent of the substrate concentration. The detection limit of the PA/NF/UR electrode was estimated to be about 0.5 µM (with S/N ) 3), which is about 1-2 orders lower than that obtained by the amperometric and potentiometric methods reported in the literature. Figure 4 shows the FIA responses obtained with the injection of solutions containing 3.0 and 1.0 µM of urea. The long-term stability of the PA/NF/UR electrode was explored by monitoring its current response to the injection of a (38) Palmer, T. Understanding Enzymes, 3rd. ed.; Ellis Horwood: New York, 1991; Chapter 6.
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Figure 5. Decay of the current response of the PA/NF/UR electrode in a 1.0 mM standard urea solution with the elapse of time. Table 2. Recovery Test for the PA/NF/UR Electrode for the Analysis of Urea in Urine Samples
Figure 4. Calibration graph obtained for the PA/NF/UR electrode in a urea concentration range from 1.0 µM to 10 mM. The inset shows the linear part of the calibration graph (a). FIA responses of the PA/ NF/UR electrode with the injection of 3.0 and 1.0 µM urea solutions (b). Experimental conditions were the same as those used in Figure 3.
1.0 mM urea standard solution at different times. Figure 5 shows the change of current responses with respect to the time elapsed. It was found that the response current decayed quickly to about 30% of its initial value after 2 weeks and became reasonably stable for the following week. The decay of sensitivity should be attributed to the denaturation of the immobilized urease and the possible leakage of urease from the cross-linking layer. The deactivation of the PA/Nafion electrode caused by the continuous reduction might be another reason for the decay of sensitivity. Interferences and Analysis of SRM and Real Samples. It was found that the PA/NF/UR electrode responds to NH4+ and has a sensitivity as large as that of urea. This should be attributed to the possible penetration of NH4+ into the urease, BSA, and GA composite layer and the following access to the PA/Nafion film. Other than NH4+ ions, the possible interferences of metallic ions, chloride ions, ascorbic acid, uric acid, and complexing agent to the urea detection were studied. Solutions containing 0.30 mM urea with the addition of 1.0 mM K+ and 20 µM each of Hg2+, 3950 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
amount of SRM added (µM)
concn found (µM)
recovery (%)
30.0 40.0 100.0 300.0
29.1 ( 0.3 39.3 ( 0.2 97.6 ( 0.5 287.5 ( 0.9
97.0 98.3 97.6 95.8
Cu2+, Pb2+, and Mg2+ were analyzed with the PA/NF/UR electrode. Compared to the results obtained with the current response of 0.30 mM standard urea solution, no appreciable change of the current response was found for solutions with added metallic ions. The response of PA/NF/UR electrode to urea analysis was not interferred by the presence of the studied ions up to the specified concentration. The inhibition effect of Hg2+ and K+ on the activity of urease reported in the literature39 was not found in this experiment. Solutions of 0.20 mM urea were added respectively with 50, 150, and 300 mM of NaCl to study the Cl- effect, and no detectable change of the current response was observed. Additions of 200 µM each of ascorbic acid and uric acid to a 50 µM urea solution were analyzed, respectively. No observable change in current response was found for the addition of these bioorganic constituents. In a similar experiment, glucose and EDTA were included, respectively, and no interference on the urea analysis was found. The PA/Nafion electrode is supposed to respond to cations in solutions.35 The partial immunity of the PA/NF/UR electrode to the metal ions studied in this experiment might be due to the screening or shielding effect of the urease, BSA, and GA coated on top of the PA/Nafion electrode. The various amino acid residues contained in the proteins of urease and BSA provide ample opportunities for the metal ions in solutions to be coordinated, intercalated, or electrostatically bound. It thus shields the (39) Ogren, L.; Johansson, G. Anal. Chim. Acta 1978, 96, 1.
Table 3. Comparison of the Performance of the Urea Biosensor Prepared from the PA/Nafion- and PPy-Modified Electrodes
electrode modifier
potential applied, E (mV vs Ag/AgCl)
linear dynamic range (µM)
polyaniline/Nafiona polypyrrole/nucleopore membraneb polypyrrolec
-200 300 -70
0.5-1000 50-1000 50-250
a
amount of urease used (units/mL) 26.4 140
Data obtained from this work. b Data quoted or estimated from Trojanowicz’s results.24
PA/Nafion electrode from the interference of metal ions or other interference in solutions. Although K+ should behave similarly to NH4+, the response of the PA/NF/UR electrode to NH4+ ions was found to be much larger than that of K+ ions in this experiment. It is not clear at present what untoward interaction in the system might be responsible for this phenomenon, and this requires further studies. To demonstrate the feasibility of the PA/NF/UR electrode for urea analysis, a NIST SRM 912A standard reference material was analyzed. The urea content of the SRM obtained agrees very well with the certified value (with a RSD of -4.61%, n ) 12). The PA/NF/UR electrode was further applied to the analysis of urea in urine samples. Because the endogeneous NH4+ in urine would interfere with the urea determination, it has to be removed prior to the analysis.40,41 In this analysis, the freshly obtained urine was filtered with a 0.45-µm membrane filter and diluted with the pH 6.50 phosphate buffer solution in a ratio of 1:2500. The diluted urine samples were then analyzed with a PA/NF/UR urea biosensor coupled HPLC. Instead of two peaks, there was only one urea peak found in the chromatograms. The absence of the NH4+ peak in the LC analysis should be due to the rather large dilution effect on the urine samples. With the standard addition method, urea contents in urine samples collected from three healthy people were found to be 264.3 ( 8.1, 603.7 ( 9.3, and 334.4 ( 6.1 mM, respectively. A recovery test for the PA/NF/ UR electrode was performed by adding various amounts of SRM to the urine samples. With four different additions of the SRM, the RSD (with n ) 4) and recoveries obtained ranged from -2.0 to -4.2% and from 95.8 to 98.0%, respectively. Table 2 shows the results. The rather high recoveries obtained confirm the feasibility of using the PA/NF/UR electrode for real sample analysis. CONCLUSIONS Compared with the similar amperometric urea biosensors reported in the literature,24,25 though the strategy of employing a (40) Smith, R. M.; Aberty, R. A. J. Am. Chem. Soc. 1956, 78, 2376. (41) Siegel, H.; Becker, K.; McCormick, D. B. Biophys. Acta 1967, 148, 655. (42) Ikariyama, Y.; Heineman, W. Anal. Chem. 1986, 58, 1803. (43) Madaras, M. B.; Spokane, R. B.; Johnson, J. M.; Woodward, J. R. Anal. Chem. 1998, 69, 3674. (44) Pankratov, O. L. J. Electroanal. Chem. 1995, 395, 35. (45) Jang, G. W.; Chen, C.; Gumbs, R. W.; Wei, Y.; Yeh, J. M. J. Electrochem. Soc. 1996, 143, 2491. (46) Wang, R.; Narang, U.; Prasad, P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671.
c
response time (s)
service life (days)
detection limit (µM)
40-120 >120 ∼300
∼21 2-3 ∼28
0.5 -50 ∼50
Data quoted or estimated from Adeloju’s results.25
conducting polymer-modified electrode as the transducer of the enzymatic products is the same, the detection mechanisms involved are different. A comparison of the performance of the urea biosensors developed in this work with that developed by Trojanowicz et al.24 and Adeloju et al.25 is shown in Table 3. A much butter performance with the developed PA/NF/UR urea biosensor is evident. In Trojanowicz’s work, the enzymatic product ammonia interacts with the modified conducting polypyrrole (PPy) during urea analysis. An irreversible process of lowering the conjugation length in the PPy chain and a buildup of CdC and NH2 groups in the polymer structure result in the deterioration of the electroactivity of PPy. It thus shortens the service life of the biosensor. It is rather surprising to find that the linear dynamic range of the urea biosensor developed by Adeloju et al. is only from 3 to 15 mg/L. Though the PPy film preparation procedure was similar to that used by Heineman et al.,42 the detection mechanism for the PPy sensor proposed by Adeloju et al. was quite different. The superiority of the proposed urea biosensor should be attributed to the adoption of a PA/Nafion electrode as the ammonium ion transducer. The close proximity of urease to the PA/Nafion electrode shortens the diffusion path of enzymatic products to the transducer and thus enhances the sensitivity of the urea biosensor and decreases the response time effectively. Combination of the PA/Nafion electrode with other deaminases, such as glutaminase, asparaginase, and creatinine iminohydrolase, should provide a simple, effective, and fast procedure for the determination of their corresponding substrates. For example, with a biosensor scheme of PA/NA/glutaminase, a glutamine sample complicated with endogeneous glutamate can be determined easily without the application of a membrane sandwich biosensor.43 Further improvement on the long-term stability of the PA/NF/UR electrode may be achieved by the incorperation of sol-gel processes44-46 into the urease immobilization procedures. ACKNOWLEDGMENT The authors thank the National Science Council of ROC for financial support of this work (Contract No. NSC 85-2113-M-110012). Received for review January 5, 1998. Accepted June 8, 1998. AC980004A
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